JP2016011849A - Method for screening preventive therapeutic agent for alzheimer's disease - Google Patents
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Abstract
Description
本発明は、アルツハイマー病予防治療薬をスクリーニングする方法に関する。 The present invention relates to a method for screening for a drug for preventing and treating Alzheimer's disease.
アルツハイマー病では、びまん性の脳萎縮、大脳皮質に老人斑（アミロイドβ（Ａβ）の沈着像）と、アルツハイマー型神経原線維変化の広範囲出現がみられる。家族性アルツハイマー病は遺伝子変異によりＡβ産生制御の異常が生じるが、孤発性アルツハイマー病の発症原因は解明されていない。 In Alzheimer's disease, diffuse brain atrophy, senile plaques (amyloid β (Aβ) deposits) in the cerebral cortex, and widespread appearance of Alzheimer-type neurofibrillary tangles are observed. In familial Alzheimer's disease, abnormalities in the control of Aβ production occur due to genetic mutation, but the cause of the onset of sporadic Alzheimer's disease has not been elucidated.
一方、エンドソームの蓄積、巨大化はダウン症やアルツハイマー病の初期にみられる現象であり、エンドソームの蓄積には、アミロイドβ前駆体蛋白質（ＡＰＰ）及びＢＡＣＥ１（(beta-site amyloid precursor protein cleaving enzyme 1)活性が必要であるが、Ａβは必要ないと報告されている（非特許文献１）。ＢＡＣＥ１産物であり、Ａβの前駆体であるＡＰＰのカルボキシ末端断片（βＣＴＦ）は、エンドソームの蓄積、巨大化を引き起こすことが報告されている。βＣＴＦのエンドソームにおける蓄積は、エンドソームの肥大化、ライソソームにおける代謝障害につながる、Traffic jam仮説が考えられているが、その分子機構はわかっていなかった。 On the other hand, the accumulation and enlargement of endosomes is a phenomenon observed in the early stages of Down's syndrome and Alzheimer's disease. The accumulation of endosomes includes amyloid β precursor protein (APP) and BACE1 ((beta-site amyloid precursor protein cleaving enzyme 1). It is reported that activity is required but Aβ is not necessary (Non-patent Document 1) A carboxy-terminal fragment (βCTF) of APP, which is a BACE1 product and a precursor of Aβ, accumulates and enlarges endosomes. The traffic jam hypothesis is considered that the accumulation of βCTF in endosomes leads to hypertrophy of endosomes and metabolic disorders in lysosomes, but the molecular mechanism was unknown.
アルツハイマー病疾患の９０％を占める孤発性アルツハイマー病の原因は明確になっておらず、その解明が待たれている。
従って、本発明の課題は、アルツハイマー病の原因解明と新たなアルツハイマー病治療薬のスクリーニング方法を提供することにある。
The cause of sporadic Alzheimer's disease, which accounts for 90% of Alzheimer's disease, has not been clarified and its elucidation is awaited.
Therefore, an object of the present invention is to provide a method for elucidating the cause of Alzheimer's disease and a screening method for a new therapeutic agent for Alzheimer's disease.
そこで、本発明者は、エンドソーム−ライソソーム機構を解明すべく、Ｐ４−ＡＴＰａｓｅファミリータンパク質と複合体をつくり、Lipid Flippaseを形成することが知られているＴＭＥＭ３０Ａ（Transmembrane protein ３０Ａ（別名ＣＤＣ５０Ａ））に着目した。ＴＭＥＭ３０Ａは、凝集Ａβと結合し、細胞膜上で凝集Ａβの受容体として機能する可能性が示唆されている（非特許文献２）が、Ａβ産生機構やアルツハイマー病発症機構に対する影響は不明であった。ＴＭＥＭ３０Ａは、Ａβ前駆体であるＡＰＰと共発現させると小胞の肥大化を誘導し、エンドソーム形態を変化させること、さらには肥大化したエンドソームは各種エンドソームが融合し、ライソソームへの成熟が阻害されていることを見出した。また、ＴＭＥＭ３０ＡはＡＰＰと結合してＡＰＰを蓄積させる作用を有すること、ＴＭＥＭ３０ＡとＡＰＰの結合部位がＢＡＣＥ１活性により産生されるＡＰＰカルボキシル末端ドメインであるβＣＴＦ領域にあることも見出した。かかる知見から、ＴＭＥＭ３０ＡとＡＰＰ又はβＣＴＦとの結合を阻害する物質を探索すれば、小胞の肥大化を抑制でき新たなアルツハイマー病予防治療薬がスクリーニングできることを見出し、本発明を完成した。 Therefore, the present inventor focused on TMEM30A (Transmembrane protein 30A (also known as CDC50A)), which is known to form a complex with P4-ATPase family protein and to form Lipid Flippase in order to elucidate the endosome-lysosome mechanism. did. It has been suggested that TMEM30A binds to aggregated Aβ and functions as a receptor for aggregated Aβ on the cell membrane (Non-patent Document 2), but the effect on Aβ production mechanism and Alzheimer's disease onset mechanism was unclear. . TMEM30A induces hypertrophy of vesicles when co-expressed with APP, an Aβ precursor, changes endosomal morphology, and the enlarged endosome fuses with various endosomes and inhibits maturation into lysosomes. I found out. It was also found that TMEM30A has an action of binding APP by accumulating APP, and that the binding site of TMEM30A and APP is in the βCTF region, which is an APP carboxyl terminal domain produced by BACE1 activity. From this finding, it was found that searching for a substance that inhibits the binding between TMEM30A and APP or βCTF can suppress the enlargement of vesicles and screen for a new preventive and therapeutic drug for Alzheimer's disease, thereby completing the present invention.
すなわち、本発明は、次の〔１〕〜〔３〕を提供するものである。 That is, the present invention provides the following [1] to [3].
〔１〕被験物質の存在下に、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの相互作用を測定することを特徴とするアルツハイマー病予防治療薬のスクリーニング方法。
〔２〕ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの相互作用が、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの結合阻害作用である〔１〕記載のスクリーニング方法。
〔３〕ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの相互作用が、培養細胞又は非ヒト動物においてＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰを共発現させて小胞体の肥大化又はＡＰＰ発現量の変化を検出するものである〔１〕記載のスクリーニング方法。
[1] A screening method for an Alzheimer's disease prophylactic or therapeutic agent, comprising measuring an interaction between TMEM30A and βCTF or APP in the presence of a test substance.
[2] The screening method according to [1], wherein the interaction between TMEM30A and βCTF or APP is a binding inhibitory action between TMEM30A and βCTF or APP.
[3] The interaction between TMEM30A and βCTF or APP allows TMEM30A and βCTF or APP to be coexpressed in cultured cells or non-human animals to detect hypertrophy of the endoplasmic reticulum or changes in APP expression [1] ] The screening method of description.
本発明方法によれば、アルツハイマー病やダウン症の初期に生じるエンドソームの肥大化を抑制するという、新たな作用機序のアルツハイマー病予防治療薬を探索できる。 According to the method of the present invention, it is possible to search for an Alzheimer's disease preventive or therapeutic drug having a new mechanism of action, which suppresses endosomal hypertrophy that occurs in the early stages of Alzheimer's disease or Down's syndrome.
ＴＭＥＭ３０Ａは、ＡＰＰと結合する作用を有し、その結合部位はβＣＴＦ中に存在する。一方、ＴＭＥＭ３０ＡとＡＰＰとを細胞中で共発現させると小胞であるエンドソームが肥大化する。このエンドソームの肥大化は、アルツハイマー病の初期に生じることである。従って、被験物質の存在下に、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの相互作用を測定し、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの結合を阻害する物質を選択すれば、アルツハイマー病の予防治療薬が探索できる。 TMEM30A has an action of binding to APP, and the binding site is present in βCTF. On the other hand, when TMEM30A and APP are co-expressed in cells, endosomes that are vesicles are enlarged. This enlargement of endosomes occurs early in Alzheimer's disease. Therefore, by measuring the interaction between TMEM30A and βCTF or APP in the presence of the test substance and selecting a substance that inhibits the binding between TMEM30A and βCTF or APP, a prophylactic or therapeutic drug for Alzheimer's disease can be searched.
ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの相互作用の測定は、in vitroでもin vivoでも行うことができる。in vitroの測定方法としては、例えば次の方法が挙げられる。
（１）ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとを含む溶液における両者の結合性と、これに被験物質を添加した場合の結合性を対比する。
ここで、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの結合性は、これらの抗体を用いた免疫沈降、ＧＳＴ融合タンパク質を用いた解析等により検出できる。
The measurement of the interaction between TMEM30A and βCTF or APP can be performed in vitro or in vivo. Examples of in vitro measurement methods include the following methods.
(1) The binding property of a solution containing TMEM30A and βCTF or APP is compared with the binding property when a test substance is added thereto.
Here, the binding property between TMEM30A and βCTF or APP can be detected by immunoprecipitation using these antibodies, analysis using GST fusion protein, or the like.
（２）被験物質の存在下で、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの共発現細胞を培養し、両者の共発現性を検出する。被験物質を添加していない場合の共発現性と対比する。このとき、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの共発現性は、これらの抗体を用いた免疫沈降、ＡＰＰの発現増強の有無等により検出できる。 (2) In the presence of a test substance, co-expressing cells of TMEM30A and βCTF or APP are cultured, and the co-expression of both is detected. Contrast with co-expression when no test substance is added. At this time, co-expression of TMEM30A and βCTF or APP can be detected by immunoprecipitation using these antibodies, presence or absence of enhanced expression of APP, and the like.
（３）被験物質の存在下で、ＴＭＥＭ３０ＡとβＣＴＦ又はＡＰＰとの共発現細胞を培養し、培養した細胞の小胞の肥大化を検出する。被験物質を添加していない場合の小胞の肥大化と対比する。 (3) In the presence of a test substance, co-expressing cells of TMEM30A and βCTF or APP are cultured, and the vesicle hypertrophy of the cultured cells is detected. Contrast with enlargement of vesicles when no test substance is added.
in vivoの測定は、被験物質を投与したアルツハイマー病モデルマウスを用いて、小胞の肥大化又はＡＰＰの発現変化を検出すればよい。被験物質を投与しないモデルマウスの小胞又はＡＰＰと対比すればよい。ここでアルツハイマー病モデルマウスとしては、Ｊ２０（家族性変異ＡＰＰ（スウェーデン型およびロンドン型の二重変異）を過剰発現するマウス）、ＴＧ２５７６(家族性変異ＡＰＰ（スウェーデン型変異）を過剰発現するマウス)等が挙げられる。 In vivo measurement may be performed by detecting vesicle hypertrophy or APP expression change using an Alzheimer's disease model mouse administered with a test substance. What is necessary is just to contrast with the vesicle or APP of the model mouse which does not administer a test substance. Here, as an Alzheimer's disease model mouse, J20 (a mouse overexpressing familial mutation APP (Swedish and London double mutation)), TG2576 (a mouse overexpressing familial mutation APP (Swedish mutation)) Etc.
次に実施例を挙げて、本発明を更に詳細に説明する。 EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.
試験例１
培養細胞にＡＰＰとＴＭＥＭ３０Ａを共発現させた。すなわち、アフリカミドリザル由来ＣＯＳ−７細胞に蛍光タンパク質Ｖｅｎｕｓをカルボキシ末端に融合させたＡＰＰ−Ｖｅｎｕｓ、及びアミノ末端に蛍光たんぱく質ｍＣｈｅｒｒｙを融合させたＴＭＥＭ３０Ａを、Ｆｕｇｅｎｅ ＨＤ（プロメガ）を用いて遺伝子導入を行った。遺伝子導入２４時間後の細胞を４％パラホルムアルデヒドを含むリン酸緩衝液で固定し、水溶性封入剤により観察用サンプルを作成した。ＡＰＰとＴＭＥＭ３０Ａの共発現は、蛍光顕微鏡により確認した（図１、Ａ）。培養細胞中の小胞の大きさと、ＡＰＰとＴＭＥＭ３０Ａの共発現を測定した（図１、Ｂ）。その結果、ＡＰＰとＴＭＥＭ３０Ａの共発現により、小胞が肥大化することが判明した（図１、Ｂ）。
Test example 1
APP and TMEM30A were co-expressed in cultured cells. Specifically, APP-Venus in which the fluorescent protein Venus was fused to the carboxy terminus of COS-7 cells derived from African green monkeys, and TMEM30A in which the fluorescent protein mCherry was fused to the amino terminus were transferred using Fugene HD (Promega). It was. Cells 24 hours after gene introduction were fixed with a phosphate buffer containing 4% paraformaldehyde, and a sample for observation was prepared using a water-soluble mounting medium. The co-expression of APP and TMEM30A was confirmed by a fluorescence microscope (FIG. 1, A). The size of vesicles in cultured cells and the co-expression of APP and TMEM30A were measured (FIG. 1, B). As a result, it was found that vesicles were enlarged by co-expression of APP and TMEM30A (FIG. 1, B).
ＣＯＳ−７細胞にＡＰＰと蛍光たんぱく質ＣＦＰをアミノ末端に融合させたＴＭＥＭ３０ＡをＦｕｇｅｎｅ ＨＤを用いて遺伝子導入した。遺伝子導入２４時間後の細胞をＡＰＰ抗体 （ＡＰＰ（Ｃ）：ＩＢＬ社）、初期エンドソームマーカーであるＲａｂ−５抗体（Ｄ−１１：サンタクルズ社）を用いて免疫染色を行った。
その結果、（図２）ＴＭＥＭ３０ＡとＡＰＰの共発現により、小胞の肥大化が確認されるとともに、Ｒａｂ−５が肥大化した小胞に集積する事が判明した、すなわちＴＭＥＭ３０ＡとＡＰＰの共発現により肥大化した小胞はエンドソームとしての性質を持つ事がわかった。
TMEM30A in which APP and the fluorescent protein CFP were fused to the amino terminus was introduced into COS-7 cells using Fugene HD. Cells 24 hours after gene transfer were immunostained using APP antibody (APP (C): IBL) and Rab-5 antibody (D-11: Santa Cruz), which is an early endosome marker.
As a result, (FIG. 2) it was found that the co-expression of TMEM30A and APP confirmed the enlargement of vesicles and that Rab-5 accumulated in the enlarged vesicles, that is, co-expression of TMEM30A and APP. It was found that the vesicles enlarged by the above have properties as endosomes.
試験例２
野生型マウスの脳と、培養細胞（ＡＰＰとＴＭＥＭ３０Ａの共発現細胞）における、ＴＭＥＭ３０ＡとＡＰＰの相互作用を検討した。すなわち、８週齢の野生型マウス脳海馬、またはＣＯＳ−７にＡＰＰとＣＦＰ融合ＴＭＥＭ３０ＡをＬｉｐｏｆｅｃｔａｍｉｎｅ２０００（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社）で遺伝子導入し、４８時間後の細胞を、１％ＣＨＡＰＳを含む溶解緩衝液（５０ｍＭ ＨＥＰＥＳ，ｐＨ７．４，１５０ｍＭ ＮａＣｌ，１ｍＭ ＥＤＴＡにプロテアーゼ阻害剤カクテルを加えたもの）で溶解し、マウス海馬はＴＭＥＭ３０Ａの抗体、培養細胞はＧＦＰ抗体（３Ｅ６ Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を用いて免疫沈降実験を行った。
その結果、マウスでも共発現細胞でも、ＴＭＥＭ３０ＡはＡＰＰと結合し、その結合はβＣＴＦ特異的であることがわかった（図３）。
Test example 2
The interaction between TMEM30A and APP in the brain of wild-type mice and cultured cells (cells that co-expressed APP and TMEM30A) was examined. Specifically, APP and CFP-fused TMEM30A were transfected into Lipofectamine 2000 (Life Technologies) into 8-week-old wild-type mouse brain hippocampus or COS-7, and 48 hours later, the cells were lysed with a lysis buffer containing 1% CHAPS ( 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA plus protease inhibitor cocktail), mouse hippocampus is TMEM30A antibody, cultured cells are GFP antibody (3E6 Life Technologies) immunoprecipitation experiment Went.
As a result, it was found that TMEM30A bound to APP in both mouse and co-expressing cells, and the binding was βCTF specific (FIG. 3).
試験例３
ＴＭＥＭ３０Ａの細胞外ドメインをＧＳＴタンパク質と融合したタンパク質を大腸菌内で発現させ、大腸菌を１％ＴｒｉｔｏｎＸ １００を含むリン酸緩衝液において溶解させた後、グルタチオンセファロースビーズを添加し、ＧＳＴ融合タンパク質をビーズに吸着させ、ビーズを３回洗浄したのち使用した。ＣＯＳ−７細胞にＡＰＰのスウエーデン型変異、または人工的なβＣＴＦであるＳＣ１００を遺伝子導入し、１％ＣＨＡＰＳを含む溶解緩衝液に溶解したのち、溶解緩衝液で平衡化した融合タンパク質の吸着したビーズを添加した。コントロールとしてＧＳＴを吸着したビーズを使用した。４℃下で１時間穏やかに攪拌を行った後、１％ＣＨＡＰＳ を含む溶解緩衝液でビーズを３回洗浄し、非結合タンパク質を除去した。ビーズに吸着したタンパク質をレムリサンプル緩衝液により溶出した。各サンプルはイムノブロット法により解析した。
その結果、βＣＴＦはＴＭＥＭ３０Ａの細胞外ドメインに直接結合することが判明した（図４）。
Test example 3
A protein in which the extracellular domain of TMEM30A is fused with the GST protein is expressed in E. coli, and the E. coli is dissolved in a phosphate buffer containing 1% Triton X 100. Then, glutathione sepharose beads are added, and the GST fusion protein is added to the beads. It was used after adsorbing and washing the beads three times. Beads adsorbed with fusion protein equilibrated with a lysis buffer after introducing SC100, which is a Swedish mutation of APP, or artificial βCTF into COS-7 cells, and lysing it in a lysis buffer containing 1% CHAPS Was added. As a control, beads adsorbed with GST were used. After gently stirring at 4 ° C. for 1 hour, the beads were washed 3 times with a lysis buffer containing 1% CHAPS to remove unbound protein. The protein adsorbed on the beads was eluted with the Remli sample buffer. Each sample was analyzed by immunoblotting.
As a result, it was found that βCTF directly binds to the extracellular domain of TMEM30A (FIG. 4).
試験例４
ＣＯＳ−７細胞にＡＰＰ単独、またはＡＰＰとＣＦＰ融合ＴＭＥＭ３０ＡをLipofectamine２０００により遺伝子導入し、遺伝子導入４８時間後のＡＰＰの代謝をイムノブロット法により解析した。ＡＰＰの代謝解析はＡＰＰのカルボキシ末端抗体（Ｃ１２Ｃ１５）もしくはβＣＴＦ特異的抗体（８２Ｅ１：ＩＢＬ社）により行った。
その結果、ＴＭＥＭ３０ＡとβＣＴＦとの共発現は、ＡＰＰ及びβＣＴＦを有意に蓄積することが判明した（図５）。
Test example 4
APP alone or APP-CFP fusion TMEM30A was introduced into COS-7 cells by Lipofectamine 2000, and APP metabolism 48 hours after gene introduction was analyzed by immunoblotting. The metabolic analysis of APP was performed using an APP carboxy terminal antibody (C12C15) or a βCTF specific antibody (82E1: IBL).
As a result, it was found that co-expression of TMEM30A and βCTF significantly accumulated APP and βCTF (FIG. 5).
試験例５
ＣＯＳ−７細胞にＡＰＰとＣＦＰ融合ＴＭＥＭ３０ＡをＦｕｇｅｎｅ ＨＤにより遺伝子導入を行い、遺伝子導入２４時間後の細胞を免疫染色により観察した。ＡＰＰカルボキシ末端抗体（ｍｃ９９（８０−９０）；ミリポア社）、後期エンドソームマーカーであるＲａｂ−７（Ｄ９５Ｆ２；Cell signaling社）、リサイクリングエンドソームマーカーであるＲａｂ−１１（Ｄ４Ｆ５；Cell signaling社）を用いた。
その結果を図６に示す。
肥大化したエンドソームは各種エンドソームマーカーが集積（通常では局在は異なる）していた（図６Ａ、Ｂ）。またLysosomeマーカー（Lysotracker:Lifetechnologies社）と一致しなかったため（図６Ｃ）、Ｌｙｓｏｓｏｍｅへの移行が阻害されている可能性を考えた。LysosomeでＡＰＰを分解するβカテプシンの阻害剤の効果を検証した結果、ＡＰＰ単独発現では阻害剤処理によりＡＰＰ−ＣＴＦが蓄積するが、ＴＭＥＭ３０Ａとの共発現でもともと蓄積したＡＰＰ−ＣＴＦは阻害剤処理により変化しない、この結果はＴＭＥＭ３０Ａとの共発現でLysosome分解低下が起こっていた事を示しており、ＡＰＰのLysosome分解が低下するとするTraffic jam仮説と矛盾しない。
Test Example 5
APP and CFP fusion TMEM30A were transfected into COS-7 cells by Fugene HD, and cells 24 hours after gene introduction were observed by immunostaining. APP carboxy terminal antibody (mc99 (80-90); Millipore), late endosome marker Rab-7 (D95F2; Cell signaling), recycling endosome marker Rab-11 (D4F5; Cell signaling) It was.
The result is shown in FIG.
In the enlarged endosome, various endosomal markers were accumulated (normally different in localization) (FIGS. 6A and 6B). Moreover, since it did not correspond with a Lysosome marker (Lysotracker: Lifetechnologies) (FIG. 6C), possibility that the transfer to Lysosome was inhibited was considered. As a result of verifying the effect of an inhibitor of β-cathepsin that degrades APP with Lysosome, APP-CTF is accumulated by treatment with an inhibitor in the expression of APP alone, but APP-CTF that has been originally accumulated by co-expression with TMEM30A is treated with an inhibitor. This result shows that Lysosome degradation was reduced by co-expression with TMEM30A, which is consistent with the Traffic jam hypothesis that Lysosome degradation of APP is reduced.
試験例６
Ａβ結合化合物であるクルクミンとタキシフォリンを用いて、ＴＭＥＭ３０ＡとβＣＴＦの結合阻害、βＣＴＦの蓄積性を検討した。まず毒性評価のため、ＣＯＳ−７細胞にクルクミン（５０、１００μＭ）、タキシフォリン（１００、２００μＭ）を加え８時間後、アラマーブルー反応液を含む培地で１時間培養し、培地に放出される蛍光物質の量をコントロール細胞（溶媒のみ加えた細胞）の値により規格化して検討した。次にＣＯＳ−７細胞にＡＰＰとＣＦＰ融合ＴＭＥＭ３０ＡをＬｉｐｏｆｅｃｔａｍｉｎｅ２０００を用いて共発現させ、２４時間後にクルクミン（５０μＭ）タキシフォリン（１００μＭ）を添加し、８時間培養した。サンプルはレムリサンプル緩衝液で溶解し、イムノブロット法により解析した。ＣＯＳ−７細胞に遺伝子導入したβＣＴＦ（ＳＣ１００）を１％ＣＨＡＰＳを含む細胞溶解緩衝液により溶解し、溶解液をクルクミン（５０μＭ）、タキシフォリン（１００μＭ）で前処理を行い、３０分後にＧＳＴ融合ＴＭＥＭ３０Ａ細胞外ドメインを吸着させたグルタチオンセファロースビーズを添加し、１時間４℃で穏やかに攪拌した。ビーズに吸着したタンパク質をレムリサンプル緩衝液により溶解してイムノブロット法により解析した
その結果、Ａβ結合化合物（図７、Ａ）は、ＴＭＥＭ３０ＡとβＣＴＦの相互作用を阻害し（図７、Ｆ）、細胞毒性が軽微な（１０％未満）濃度で（図７、Ｂ）ＡＰＰ−βＣＴＦの蓄積を減少させた（図７Ｃ〜Ｅ）。
Test Example 6
The inhibition of binding between TMEM30A and βCTF and the accumulation of βCTF were examined using curcumin and taxifolin, which are Aβ binding compounds. First, for toxicity evaluation, curcumin (50, 100 μM) and taxifolin (100, 200 μM) were added to COS-7 cells, and after 8 hours, the cells were cultured for 1 hour in a medium containing an alamar blue reaction solution. The amount of the substance was standardized by the value of control cells (cells added with solvent only) and examined. Next, APP and CFP-fused TMEM30A were co-expressed in COS-7 cells using Lipofectamine 2000, and curcumin (50 μM) taxifolin (100 μM) was added 24 hours later and cultured for 8 hours. Samples were lysed with Laemmli sample buffer and analyzed by immunoblotting. ΒCTF (SC100) gene-introduced into COS-7 cells was lysed with a cell lysis buffer containing 1% CHAPS, and the lysate was pretreated with curcumin (50 μM) and taxifolin (100 μM), and after 30 minutes, GST-fused TMEM30A Glutathione sepharose beads adsorbed with extracellular domain were added and gently stirred at 4 ° C. for 1 hour. The protein adsorbed on the beads was dissolved in the Remli sample buffer and analyzed by immunoblotting. As a result, the Aβ binding compound (FIG. 7, A) inhibited the interaction between TMEM30A and βCTF (FIG. 7, F). Concentration of APP-βCTF was reduced (FIGS. 7C-E) at concentrations with minimal cytotoxicity (less than 10%) (FIG. 7, B).
試験例７
クルクミンとタキシフォリンが、ＴＭＥＭ３０ＡとＡＰＰ共発現系に及ぼす作用について検討した。ＣＯＳ−７細胞にＶｅｎｕｓ融合ＡＰＰ、及びｍｃｈｅｒｒｙ融合ＴＭＥＭ３０ＡをＦｕｇｅｎｅ ＨＤにより遺伝子導入した。遺伝子導入２４時間後、クルクミン（５０μＭ）、タキシフォリン（１００μＭ）を含む培地で８時間培養した。
その結果、ＴＭＥＭ３０ＡとＡＰＰの共発現によりＡＰＰ発現蛍光強度が強くなるが、クルクミンとタキシフォリンは、いずれも、その蛍光強度を低下させた（図８）。
Test Example 7
The effects of curcumin and taxifolin on TMEM30A and APP co-expression system were examined. Venus-fused APP and mcherry-fused TMEM30A were introduced into COS-7 cells by Fugene HD. 24 hours after gene introduction, the cells were cultured for 8 hours in a medium containing curcumin (50 μM) and taxifolin (100 μM).
As a result, the APP-expressed fluorescence intensity increased due to co-expression of TMEM30A and APP, but both curcumin and taxifolin decreased the fluorescence intensity (FIG. 8).
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